Blood comprises hematopoietic cells such as erythrocytes, leukocytes, platelets and the like. These hematopoietic cells mature from only one kind of pluripotential blood stem cell through various differentiation steps. These steps undergo complex regulation by a group of proteinous factors which are generally referred to as cytokines. A certain type of cytokine takes part in the differentiation and multiplication of various hematopoietic cells. On the other hand, a certain type of hematopoietic cell undergoes regulation of its differentiation and multiplication by various types of cytokines. This is called overlapping cytokine actions. Among these cytokine members, TPO and G-CSF are considered to have small overlapping actions.
TPO mainly takes part in the formation of platelets. Platelets are formed by the fragmentation of megakaryocytes, a hematopoietic cell which has a large nucleus and is present mainly in bone marrow. Platelets are essential for forming blood clots at damaged portions in blood vessels. Platelets also play important roles in not only blood coagulation but also injury healing by releasing proteins having other functions at the damaged portions. A significant decrease in the number of platelets may be fatal, because the body may easily bleed.
G-CSF is a cytokine which accelerates activation of neutrophils, a member of the leukocytes, and differentiation of neutrophils from their precursor cells. Neutrophils exert the first defense action when invaded by foreign enemies such as bacteria, viruses and the like. When the number of neutrophils is decreased, the body becomes defenseless against infection, and this too is also often fatal.
Current medical treatment of cancers often cause side effects in which pluripotential blood stem cells are damaged by the administration of a chemotherapeutic drug, irradiation of X-rays or bone marrow transplantation for the treatment of leukemia, thus decreasing the number of all hematopoietic cells. Apparently, it is markedly beneficial for thrombopenia and leukopenia patients to amplify the number of these cells by the administration of cytokine, to suppress bleeding tendency and preventing infectious diseases.
A cytokine which can amplify platelets and neutrophils simultaneously has not been found, and there is no medicine having such an effect.
Leukemia inhibiting factors, stem cell factors, macrophage colony stimulating factors, granulocyte/macrophage colony stimulating factors, erythropoietin, interleukin (IL)-3, IL-6, IL-11, megakaryocyte colony stimulating factors and the like are known as substances which amplify platelets or enhance differentiation and multiplication of megakaryocytes [Metcalf et al., Blood, 80, 50-56 (1990); Hunt et al., Blood, 80, 904-911 (1992); Examined Japanese Patent Publication No. 6-11705; Hoffman et al., Blood Cells, 13, 75-86 (1987); Mazur et al., Exp. Hematol., 15, 1123-1133 (1987); McNiece et al., Exp. Hematol., 16, 807-810 (1988); Lu et al., Brit. J. Hematol., 70; 149-156 (1988); Ishibashi et al., Proc. Natl. Acad. Sci. USA, 86, 5953-5957 (1989); WO 95/21919; WO 95/18858]. It is understood that these many cytokine members amplify platelets by overlapping actions. Recently, it was revealed that a receptor ligand called c-mpl is a cytokine which has the highest activity among platelet amplifying factors and acts directly [de Sauvage et al., Nature, 369, 533 (1994)].
As substances which multiply granulocytes, the above-mentioned IL-3, macrophage colony stimulating factors, granulocyte/macrophage colony stimulating factors and the like are known, but G-CSF has the highest activity in terms of multiplying neutrophils selectively [Nicola et al., J. Biol. Chem., 258, 9017 (1983)]. With regard to a polypeptide in which two different kinds of cytokine are fused, there are reports in Japanese Published Unexamined International Patent Application No. 500116/94, U.S. Pat. No. 5,359,035, Exp. Hematol., 21, 647-655 (1993) and ibid., 18, 615 (1990) and the like.
However, nothing is known about a fusion polypeptide in which TPO is used as one of the fused cytokines.
An object of the present invention is to provide a fusion polypeptide which can produce and amplify platelets and neutrophils simultaneously. This fusion polypeptide allows the formation of megakaryocyte colonies and neutrophil colonies and the differentiation or maturation of megakaryocyte precursor and neutrophil precursor can be controlled.